1. Field of the Invention
The present invention relates generally to the gene, and mutations, that are responsible for the disease hemochromatosis (MIM604653). In particular, the present invention provides for the presence of one or more mutations on the SLC11A3 gene which results in aberrant SLC11A3 mediated iron transport. The invention also relates to methods for screening for HH and to HH diagnosis, prenatal screening and diagnosis, and therapies of HH disease, including gene therapeutics, protein and antibody based therapeutics, and small molecule therapeutics. The invention further relates to drugs and therapies developed for the treatment of patients with HH or anemia.
2. Description of the Related Art
Over the years, several known genes involved in iron metabolism have been implicated in the pathology of HH. However, not all instances of HH patients can be explained by mutations in these genes. In particular, it is known that approximately 60-85% of all instances of HH in adult patients are indicated by homozygosity for the C282Y mutation in the HLA-H/HFE gene on Chromosome 6p. Compound heterozygosity accounts for an additional 10% of cases. It is also known that a form of juvenile hemochromatosis maps to chromosome 1q and that a single family was found with a mutation in a transferrin receptor gene (TFR2) on chromosome 7q. However, the remaining 5-15% of patients indicated with HH do not possess any mutations on the known genes. For example, Kato et al., describe a heterzygous A49T mutation in the 5_UTR of the H-subunit of ferritin without specifically delineating the contributions of this gene to the HH disease state (Am. J. Human Genetics 69: 191-7 (2001)). Clearly, neither the precise physiological mechanism of iron overaccumulation nor every gene which is defective in this disease has been described.
Hemochromatosis is an inherited disorder of iron metabolism wherein the body accumulates excess iron. In symptomatic individuals, excess iron is deposited in a variety of organs which leads to organ failure. Disease states such as cirrhosis, diabetes, sterility, and other serious illnesses occur as a result. It has also been discovered that HH can be inherited by a dominant or pseudo-dominant mode of inheritance. Heretofore, HH was believed to be inherited solely as a recessive trait. In particular, the prior art limits HH to patients having homozygotes carrying two defective copies of the gene.
Hemochromatosis is also one of the most common genetic disorders. The prior art estimates that approximately 10% of individuals of Western European descent carry one copy of the HH gene mutation and that there are about one million homozygotes in the United States. HH, thus, represents one of the most common genetic disease mutations in individuals of Western European descent. Although HH ultimately produces debilitating symptoms, the majority of carriers have not been diagnosed. Indeed, it has been estimated that no more than a small fraction of affected individuals in the United States have been diagnosed with this condition.
Current diagnostic methods fail to comprehensively test for HH in individuals who are at risk, especially those individuals who are presymptomatic. Although blood iron parameters can be used as a screening tool, a confirmed diagnosis often employs a costly, invasive and high risk liver biopsy. An additional problem is that symptoms of HH are similar to those of other conditions and the severe effects of the disease tend not to appear immediately. Thus, there is a clear need for the development of an inexpensive and noninvasive comprehensive diagnostic test for detection of HH in order to facilitate comprehensive diagnosis and to provide comprehensive presymptomatic detection for the identification of HH carriers. Accordingly, comprehensive methods to identify persons who may be destined to become symptomatic in order to intervene in time to prevent excessive tissue damage associated with iron overload are desirable.
U.S. Pat. No. 6,025,130 (xe2x80x9cThomas et al.xe2x80x9d) describes a gene indicated for HH in the HLA region of Chromosome 6. Although Thomas et al. discloses a gene thought to cause HH, it does not provide for the remaining population of individuals who are symptomatic of HH but do not carry the mutation as taught by Thomas et al.
In the search for additional genes indicated for HH, one study generally concluded that HH can occur in adults who do not have pathogenic mutations in the hemochromatosis gene. See N Engl J Med 341:725-32 (1999). In particular, the study found that members of a single family of Italian descent displayed symptoms of one or more distinct genetic diseases that cause a type of adult hereditary iron overload which was not associated with the known HH gene. The study, however, failed to provide for the gene hypothesized to cause the disease.
Another study relating to Zebrafish concluded that the gene ferroportin 1 (FNP1) may be involved in mammalian disorders of iron deficiency or overload. Nature, 403:17 (2000). In particular, the study focused on hypochromia caused by inadequate circulatory iron levels in embryonic Zebrafish. The study found that two independent autosomal recessive mutations caused hypochromia in the Zebrafish embryo. Mammalian homologues of these genes include SLC11A3.
However, the transversion site disclosed by the study differs from the present invention and is not believed to be responsible for HH. In particular, the study specifically teaches that a C-to-A transversion at codon 361 prematurely terminates translation of the SLC11A3 gene and that a G-to-T transversion at an undisclosed location results in a single amino acid change from Leucine to Phenylalanine. Additionally, the study fails to explain how the amino acid change affects the SLC11A3 protein. Accordingly, the study does not provide any motivation or suggestion to one of ordinary skill in the art to believe that a C-to-A transversion at codon 361 or a G-to-T transversion at an undisclosed location causes about 5-15% of all HH cases.
Genetic markers are also known for a mutation in recessive hemochromatosis. Diagnostic methods and kits for its determination are disclosed by U.S. Pat. Nos. 5,753,438 and 5,705,343.
U.S. Pat. No. 5,712,098 (xe2x80x9cTsuchibashi et al.xe2x80x9d) also discloses recessive hereditary hemochromatosis diagnostic markers and diagnostic methods for same.
U.S. Pat. No. 5,674,681 (xe2x80x9cRothenbergxe2x80x9d) discloses a method for identifying an individual having or predisposed to having hemochromatosis by detecting a mutation in the gene encoding a nonclassical MHC class I heavy chain. Rothenberg also discloses methods for treating recessive hemochromatosis involving administration of a xe2x80x9cleczymexe2x80x9d having similar specificity for a carbohydrate ligand as the leczyme involved in the disease state. Clearly, the prior art fails to provide for the remaining 5-15% of patients indicated with HH who do not possess any mutations of known genes.
Accordingly, it would be highly desirable to identify, isolate, clone, and sequence the gene responsible for the remaining 5-15% of patients indicated with HH who do not possess any mutations of known genes. Such identification, isolation, cloning, and sequencing of the gene would enable the design and manufacture of products useful for the diagnosis and screening for HH. Identification of individuals affected with HH will allow initiation of this therapy, which can prevent symptoms, arrest progression of organ damage, and in some cases reverse pathology due to iron overload.
Moreover, such identification, isolation, and cloning of the gene would enable the study of the operation of the gene in the development of iron overload diseases, in general, and HH in particular. Further, it would be highly desirable to provide therapeutics for iron overload diseases, and HH disease in particular, as well as oxidative free radical diseases, reactions, and processes in general. The identification, isolation, sequencing, and cloning of the gene responsible for the remaining 5-15% of patients indicated with HH who do not possess any mutations of known genes, and identification of its protein products would also facilitate improved therapeutic development.
These and other embodiments of the invention will be apparent from the detailed description and the claims.
The present inventive subject matter relates to an identified, isolated, cloned and sequenced mutated SLC11A3 gene responsible for HH in about 5-15% of patients indicated with HH who do not possess any mutations of known genes thought to cause HH.
The principal mutation found in the gene comprises a single nucleotide substitution of A-to-C at positions 734 (A734C) in exon 5 which causes a significant amino acid change in the protein product expressed by the gene. One such amino acid change that may cause disruption of normal regulation of SLC11A3 activity as a result is the amino acid substitution of asparagine by histidine at position 144 (N144H).
The present invention, therefore, represents the first opportunity to accurately and noninvasively screen and diagnose HH in the 5-15% of patients indicated with HH who do not possess any mutations of known genes thought to cause HH. In addition, the present invention enables the study of the SLC11A3 gene. Through such study, the development of therapeutics (gene, protein replacement, antibodies, small molecules, and the like) for HH disease will be enabled.
In accordance with a first embodiment of the present invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO: 1.
Another embodiment of the present inventive subject matter is a method for diagnosing a patient as having an increased risk of developing HH disease, comprising the steps of providing DNA or RNA from the individual; and assessing the DNA or RNA for the presence or absence of a base mutation at position 734 (A734C) in exon 5 of the SLC11A3 gene wherein the absence of the base mutation indicates the absence of a HH gene mutation in the genome of the individual and the presence of the base mutation indicates the presence of the HH gene mutation in the genome of the individual and an increase risk of developing HH disease.
Yet another embodiment of the present inventive subject matter is an oligonucleotide in a range of 8-18 consecutive nucleotides selected from a sequence unique to SEQ ID NO: 1 or the complement of SEQ ID NO: 1.
Another embodiment of the present inventive subject matter is a isolated polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, wherein SEQ ID NO: 2, SEQ ID NO: 3, and are the normal peptide product of the SLC11A3 gene, the mutated peptide product of the SLC11A3 gene and the wild type peptide product of the SLC11A3 gene, respectively.
Yet another embodiment of the inventive subject matter is a pharmaceutical composition comprising the isolated polypeptide as shown in SEQ ID NO: 2.
Another embodiment of the present inventive subject matter is a kit for the detection of the presence or absence of a base mutation at position 734 (A734C) of the SLC11A3 gene. In another embodiment, the kit further comprises oligonucleotide primers for amplifying the DNA containing the base-pair polymorphisms.
Yet another embodiment of the present inventive subject matter is a genetic marker predictive of a hereditary hemochromatosis (HH) gene mutation comprising a partial sequence of SEQ ID NO: 1 and sequences complementary therewith. In one aspect, the nucleic acid is DNA. In another aspect, the DNA is cDNA. In another aspect, the nucleic acid is RNA. In another aspect, the nucleic acid is a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO: 1.
Still yet another embodiment of the present inventive subject matter of the invention is a cloning vector comprising a coding sequence of a nucleic acid as set forth above and a replicon operative in a host cell for the vector.
Further still yet another embodiment of the present inventive subject matter is an expression vector comprising a coding sequence of a nucleic acid set forth above operably linked with a promoter sequence capable of directing expression of the coding sequence in host cells for the vector.
Another embodiment of the present inventive subject matter is host cells transformed with a vector as set forth above.
Yet another embodiment of the present inventive subject matter is a method of producing a mutant SLC11A3 polypeptide comprising: transforming host cells with a vector capable of expressing a polypeptide from a nucleic acid sequence as set forth above; culturing the cells under conditions suitable for production of the polypeptide; and recovering the polypeptide.
Still yet another embodiment of the present inventive subject matter is a peptide product consisting of a polypeptide having the amino acid sequence corresponding to the sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In one aspect, the peptide is labeled. In another aspect, the peptide is a fusion protein.
Further still yet another embodiment of the present inventive subject matter of the invention is a use of a peptide as set forth above as an immunogen for the production of antibodies. In one embodiment, there is provided an antibody produced in such application. In one embodiment, the antibody is labeled. In another embodiment, the antibody is bound to a solid support. In a further embodiment, the antibody is monoclonal. Another embodiment of the present inventive subject matter is an oligonucleotide primer useful for amplification of DNA, the oligonucleotide primer designed on the basis of the DNA sequence of any one of SEQ ID NO: 1.
Yet another embodiment of the present inventive subject matter is a method for diagnosing whether a patient is afflicted with hereditary hemochromatosis (HH) disease, comprising: contacting cells of the patient with antibodies directed against an epitope on an SLC11A3 protein product corresponding substantially to SEQ ID NO: 3, or SEQ ID NO: 4; and observing whether the antibodies localize on the cells. In one embodiment, the method is conducted in vitro. In another embodiment, the method is conducted in vivo.
Still yet another embodiment of the present inventive subject matter is a method for treating a patient diagnosed as having hereditary hemochromatosis (HH) disease, comprising delivering a polypeptide corresponding to the amino acid sequence of SEQ ID NO: 2 to tissues of the patient. In one aspect, the polypeptide is delivered directly to the tissues. In another aspect, the polypeptide is delivered intravenously. In another embodiment, the polypeptide is delivered to the tissues through gene therapy.
Further still yet another embodiment of the present inventive subject matter is an animal model for hereditary hemochromatosis (HH) disease, comprising a mammal possessing a mutant or knocked-out SLC11A3 gene.
Another embodiment of the present inventive subject matter is metal chelation agents derived from nucleic acid sequences described above or from a peptide product as described above in a physiologically acceptable carrier. In one embodiment, the metal is selected from the group consisting of iron, mercury, cadmium, lead, and zinc.
Yet another embodiment of the present inventive subject matter is a method to screen mammals for susceptibility to metal toxicities, comprising, screening such mammals for a mutation in the SLC11A3 gene and wherein those mammals identified as having a mutation are more susceptible to metal toxicities than mammals not identified as having a mutation. In one aspect, the metal is selected from the group consisting of iron, mercury, cadmium, lead, and zinc.
Still yet another embodiment of the present inventive subject matter is a method for selecting patients infected with hepatitis virus for xcex1-interferon treatment, comprising screening such patients for a mutation in the HH gene and wherein those patients not identified as having a mutation are selected to proceed with xcex1-interferon treatment and those identified as having a mutation are selected to undergo phlebotomy prior to xcex1-interferon treatment.
Further still yet another embodiment of the present inventive subject matter is a T-cell differentiation factor comprising a moiety selected from the group consisting of molecules derived from nucleic acid sequences described above and from peptide products described above.
Another embodiment of the present inventive subject matter is a method for screening potential therapeutic agents for activity in connection with HH disease, comprising: providing a screening tool selected from the group consisting of a cell line, a cell free, and a mammal containing or expressing a defective SLC11A3 gene or gene product; contacting the screening tool with the potential therapeutic agent; and assaying the screening tool for an activity selected from the group consisting of SLC11A3 protein folding, iron uptake, iron transport, iron metabolism, receptor-like activities, upstream processes, downstream processes, gene transcription, and signaling events.
Yet another embodiment of the present inventive subject matter is a therapeutic agent for the mitigation of injury due to oxidative processes in vivo, comprising a moiety selected from the group consisting of molecules derived from nucleic acid sequences described above and from peptide products described above.
Yet another embodiment of the present inventive subject matter is a method for diagnosing whether a patient is afflicted with anemia, comprising: contacting cells of the patient with antibodies directed against an epitope on an SLCllA3 protein product corresponding substantially to SEQ ID NO: 3, or SEQ ID NO: 4; and observing whether the antibodies localize on the cells. In one embodiment, the method is conducted in vitro. In another embodiment, the method is conducted in vivo.
Further still yet another embodiment of the present invention involves a kit for detecting or identifying HH or anaemia in a sample, comprising a means for collecting a sample of breast epithelial cells and a means for detecting an A-to-C transversion at position 734 (A734C) in the SLC11A3 gene.
Yet another embodiment of the present invention is directed to a pharmaceutical composition for treating iron overload or anaemia in an animal, comprising a therapeutically effective amount of an catalytically active fragment of an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and a pharmaceutically acceptable carrier.